Abstract

A pyrimidine cluster analysis of the light strand of HeLa mitochondrial DNA has been undertaken. The major difficulties in this attempt to obtain the pyrimidine oligonucleotide distribution in mitochondrial DNA are the limited amount of material available and the multistep procedure which may incur losses of DNA material as well as complications leading to irreproducible results.
To overcome the scarcity of material, the sensitivity of isotopic labelling is enhanced by using the enzyme 5'-OH-polynucleotide kinase to attach a radioactive phosphate to the end of each oligonucleotide. The fingerprinting procedure is adapted to the microgram level of DNA material by thin layer displacement chromatography.
The complications involved by multiple enzyme reactions have been solved by varying one parameter at a time. As a result of eight experiments, the conditions for a successful pyrimidine oligonucleotide analysis have been outlined.